Effect of Puccinia silphii on Yield Components and Leaf ...the domestication of wild plants has been...

sustainability

Article

Effect of Puccinia silphii on Yield Components andLeaf Physiology in Silphium integrifolium Lessonsfor the Domestication of a Perennial Oilseed Crop

M Kathryn Turner 1 Damian Ravetta 2 and David Van Tassel 1 ID

1 The Land Institute 2440 E Water Well Rd Salina KS 67401 USA turnerlandinstituteorg2 National Scientific and Technical Research Council (MEF-CONICET) Fontana 140 Trelew 26035 Argentina

ravettaagroubaar Correspondence vantassellandinstituteorg Tel +1-(785)-823-5376

Received 10 February 2018 Accepted 1 March 2018 Published 5 March 2018

Abstract New crops with greater capacity for delivering ecosystem services are needed to increaseagricultural sustainability However even in these crops seed yield is usually the main criteria forgrain domestication This focus on yield can cause unintended structural and functional changesLeaves of selected plants tend to be more vulnerable to infection which can reduce performanceassimilates and ultimately yield Our objectives were to determine the impact of rust (causedby Puccinia silphii) on yield and leaf function in selected Silphium integrifolium (Asteraceae) plantsWe tested the effect of a fungicide treatment on rust severity and yield compared the rust infection ofindividuals in a population selected for yield and related this to chemical changes at the leaf levelWe also estimated heritability for rust resistance We found that productivity indicators (head numberand weight leaf weight) and leaf processes (photosynthetic capacity water use efficiency) werereduced when silphium leaves and stems were more heavily infected by P silphii Leaf resin contentincreased when susceptible plants were infected Fungicide treatments were effective at reducing rustinfection severity but were ineffective at preventing yield losses We propose that disease resistanceshould be included early in the selection process of new perennial crops

Keywords disease control new crops rust resin photosynthesis gas exchange

1 Introduction

Although diseases and herbivory are key factors in determining yield in crops the process ofthe domestication of wild plants has been generally analyzed in terms of yield and yield componentsand less in terms of specific disease resistance or the potential factors contributing to the resistanceDuring early stages of domestication in seed crops disease resistance tends to be captured only as itaffects seed yield and potentially persistence in perennial crops Unintended structural and functionalchanges can occur together with yield increase and there is recent evidence of anatomical andfunctional changes at the leaf level that occur very early in the domestication process [1] Structural andfunctional changes resulting from the selection for yield such a leaf thickness cell size and secondarymetabolites (ie resins) are traits known to influence the response to pathogens [2ndash4] Among a groupof new perennial grain crops under domestication Silphium integrifolium Michx (silflower silphiumrosinweed) has been selected as a new dual use oilseed and biomass crop [5] Silphium is native tocentral and eastern North America and was selected for domestication primarily due to its droughttolerance and high seed oil content

Rust caused by Puccinia silphii [6] is likely to be an important disease in the development ofsilphium Puccinia silphii is a microcyclic fungal pathogen producing only the hyaline basidiosporeand over-wintering teliospore and lacking the haploid pychniospore dikaryotic aeciospore and the

Sustainability 2018 10 696 doi103390su10030696 wwwmdpicomjournalsustainability

Sustainability 2018 10 696 2 of 12

long-distance dispersing urediniospore stages of macrocyclic rusts [7] It has been the most prevalentdisease in introductory plantings and breeding plots in central Kansas although powdery mildew(caused by Erysiphe cichoracearum) downy mildew (caused by Plasmopara halstedii) and stem spotting(caused by Alternaria alternate) have also been observed Between 2003 and 2013 there were very fewpests or diseases observed in silphium research plantings in Kansas The low initial levels of rustincidence may have been due to limited local sources of inoculum when the populations of silphiumwere small in research plantings and inoculum was spreading from dispersed native stands In 2014high moisture promoted the spread of rust from a densely planting stand of silphium to many ofthe other plots By 2015 many silphium plants failed to flower or produced very small late flowersSome of these individuals had necrosis on the apical meristem or entire length of the stalk and othershad severely stunted plant height These symptoms have persisted in subsequent years and promptedinterest in determining the impact of rust on the yield and physiology of silphium

Rust-causing pathogens are detrimental to biomass production and grain yield acrossmany economically significant crops Puccinia triticina Eriks which causes leaf rust in wheat(Triticum aestivum) durum wheat (T turgidum L var durum) cultivated emmer wheat (T dicoccon)and wild emmer wheat (T dicoccoides) Aegilops speltoides goatgrass (Ae cylindrica) and triticale(X Triticosecale) [8] reduces the photosynthetic capacity of the flag leaf and lower leaves as it utilizesassimilates that would be translocated to developing grain resulting in lower seed set and smallerkernel weight [8] The quality of the grain can also be affected Decreased protein content andincreased yellow pigmentation have been observed in infected grain [8ndash10] but vary by genotype andenvironment [11] Changes in photosynthesis due to foliar pathogens have been reported across a rangeof crop species including leaf rust (Puccinia hordei Otth) in barley (Hordeum vulgare L) [12] powderymildew (Eysiphe polygoni DC) in sugar beet (Beta vulgaris L) [13] and rust (Uromyces appendiculatus)angular leaf spot (Phaeoisariopsis griseola (Sacc) Ferr) and anthracnose (Colletotrichum lindemuthianum)in common bean (Phaseolus vulgaris L) [14] Besides reduction in photosynthesis infections cancause other physiological changes such as reduced water use efficiency as observed in powderymildew (Uncinula necator) in grape (Vitis vinefera and V labruscana) [15] and powdery mildew(Puccinia lagenophorae) in groundsel (Senecio vulgaris) under drought conditions [16]

Our objectives here were to determine the impact of silphium rust on plant yield and leaf functionWe compared the rust infection of individuals in a population of plants selected for yield and relatedthis to chemical changes at the leaf level We also estimated heritability for rust resistance and testedthe effect of a fungicide treatment on rust severity and yield

2 Materials and Methods

21 Susceptible and Resistant Plant Materials

We adapted the Puccinia helianthus disease scales developed by Gulya (1990) [17] and Sackston(1962) [18] to quantify P silphii percent severity (proportion of the total leaf area infected) and response(a measure of pustule size and vigor) on the leaves and stems of silphium plants The pictorial scalesfor severity and response (Supplementary Materials Figure S1) were used in July 2015 to identifyindividuals with high and low disease levels which were used as susceptible and resistant checksrespectively The determination of resistance and susceptibility was based on our observations ofinfection caused by naturally occurring pathogen populations in our breeding nurseries We arenot aware of any description of P silphii races differential reaction to various isolates or silphiumgenotypes evaluated for resistance to P silphii Resistant plants (le1 severity of infection on the leaves)included SI1100 SI1066 and SI1830 Susceptible plants (gt2 leaf severity) included SI591 SI1619and SI175 In 2015 very few flowering heads were produced by SI1066 SI1830 SI1619 and SI175These individual genets were identified from a large breeding nursery of over 7000 genets from100 half-sib families planted approximately 1 m from each other in 2013 Rust symptoms were seenon the edges of this plot in 2014 and by 2015 the vast majority of plants in the nursery were infected


Check genets were replicated by digging and dividing crowns in December 2015 Cloned rhizomeswere placed in pots containing soilless potting medium and allowed to regrow in a greenhouse priorto transplanting in the spring of 2016

22 Fungicide Experiment

To test whether fungal pathogens P silphii E cichoracearum and P halstedii were causingnecrosis and yield losses we designed a randomized block trial in a field planted in 2014 thathad experienced severe stalk necrosis and natural infection by rust powdery mildew and downymildew observed in 2015 Each block contained 15 existing randomly selected individuals and eightreplicated checks with three reps of each treatment inoculum was produced by natural infectionThe checks included two Helianthus annuus accessions from the USDArsquos (United States Departmentof Agriculture) National Plant Germplasm System (HA-89 and HA-292) three resistant and threesusceptible Silphium integrifolium genets from the checks described above H annuus accessionswere planted from seed and S integrefolium genets were transplanted from clones on 26 April 2016Two treatments fungicide and water control were replicated three times and applied every 3 weeksthrough flowering following the manufacturerrsquos application rate for Puccinia species (12 gm2)The water applied to the control treatment was equal in volume to the fungicide application No watertreatment was applied at the time of the initial fungicide application Azoxystrobin (Heritagereg with theactive ingredient methyl (E)-2-2-[6-(2-cyanophenoxy)pyrimidin-4-yloxy]phenyl-3-methoxyacrylate)was selected based on its recommended use in controlling rust in H annuus production [19] as well aspowdery and downy mildews

The measurements of rust percent severity of infection and response (pustule size and vigor)on the leaves (Supplementary Materials Figure S1) were recorded based on visual observations onthree dates in the summer at least one week after fungicide or water application Clonal checks weresubsequently used to calculate heritability on an entry mean basis as described by Fehr (1987) [20]The number of stalks per plant stalks with complete necrosis or necrosis of the meristem and thenumber of capitula per stalk were counted on one date between the stages of seed fill and senescenceAt maturity three stalks per plant were harvested and weighed The capitula leaves and stems froma subset of plants were dried to obtain the mean moisture content of each organ The fresh weightswere multiplied by the coefficient 1mdashpercent moisture to estimate dry weight Biomass from theleaves (n = 57 plants) stems (n = 57) and capitula (including seeds and the receptacle n = 173) wereweighed separately

Prior to flowering in June 2016 we measured gas exchange at the leaf level on a subset of theexperimental entries with 01 05 1 2 and 5 severity of the leaf area infected Each severity classwas represented by measuring three fully expanded leaves from six plants except the uncommon5 disease severity class which was only represented by two plants To measure Net CO2 uptake(A) and transpiration (E) we used a portable- infrared gas analyzer (LCA-4 ADC Hogdesdon UK)We measured gas exchange on fully developed leaves of same age (last fully developed leaf on a stalk)We recorded the severity of rust infection in each of the leaves on which gas exchange was measuredLeaves were exposed to full radiation and measurements were done on a sunny day between 11 AMand 2 PM to avoid environmental self-shading and provide reliable comparable estimations of gasexchange Water use efficiency (WUE) was estimated by dividing net CO2 uptake by transpirationUnless specified all results represent the experimental lines and do not include the checks

23 Leaf Resin Content

In August 2017 we identified several plants in a breeding nursery (established from seedlingsin the spring of 2015) that typified either strong or weak partial-resistance to rust Leaves wereharvested from individual plants dried and ground for resin extraction We harvested infectedand uninfected leaves both on plants that had a low level of infection (resistant) and a high level ofinfection (susceptible) which was defined based on the severity of infection on the whole plant based


on natural infection Plants were considered susceptible if more than 50 of the leaves were infectedat a severity of ge1 and resistant if rust was present on less than 20 of the leaves at a severityof lt1 Leaf resin was extracted using dicloromethane (DCM) in a Soxhlet apparatus for 6 h untilexhaustion [21] DCM extracts were weighed and resin content was estimated on a dry-weight basisThe term ldquoresinrdquo is used to describe a complex mixture of non-polar secondary metabolites mostly butnot uniquely terpenes (sesqui- and diterpenes) [22] They are extracted with non-polar organic solventssuch as dicloromethane Resins are common in many species of Asteraceae thus the common namerosinweed of several species in the family

Treatment differences were tested using an ANOVA and Tukeyrsquos test for mean separation fromthe R package lsquolaerciorsquo and correlations were tested using the Pearsonrsquos correlation coefficient in the Rbase package [23]

3 Results and Discussion

We found a range of rust infection severity in silphium plots growing in Central Kansasfrom 0ndash20 infection Rust severity increased through the growing season and was highest inJune (Figure 1a) The peak level of disease occurring in June may have been due to a reductionin relative humidity and rainfall which are important to rust disease development [24] causing leavesformed later in the growing season to have lower levels of infection The fungicide-treated plots weresignificantly lower in rust disease severity in May and June but were not significantly different inJuly (Figure 1a) Only minimal control of infection was attained with fungicide As the plants grewthrough the season and the canopy became denser it was not possible to obtain complete fungicidecoverage which may have accounted for the decrease in control efficiency throughout the seasonThe water control was applied because higher moisture especially in the form of light mist as in thefungicide application could promote spore germination and higher disease levels High humidityis required and is the most important factor for basidiospore germination basidiospores are verysensitive to desiccation caused by low humidity or direct sunlight germination can also be inhibitedby free water [24] However the absence of the first water control treatment did not prevent significanttreatment differences in the first ratings in May and even with lower moisture the control plots hadhigher infection (Figure 1a) Powdery and downy mildew infection was minimal including in thecontrol plots and was thus not recorded

The H annuus accessions were not infected with rust during the trial (Figure 1b) The host rangeof P silphii is highly specialized and limited to the Silphium genus [25] The silphium cloned checksexhibited a large range of infection from 0 to 17 average severity Although treatment differenceswere not significant probably due to the small number of treatment replicates (three) for each clonedcheck generally the fungicide-treated plot appeared to have lower infection levels even for the moreresistant genets (Figure 1b) These results indicate that a high amount of variation is available forselection within the breeding populations and that fungicide may be able to modestly decrease theseverity of infection even in resistant genets SI1066 was selected as a resistant individual in 2015but in 2016 had higher infection rates between 2 and 10 severity in two of the control treatmentplots In 2015 this individual had the higher infection level compared to the other resistant checks(1 severity) but may have partially escaped severe rust infection due to some other disease or insectpressure that also prevented it from flowering or its most heavily infected leaves may have been lostto senescence by the time of selection in July

We found the heritability of percent severity to be moderately high The heritability on an entrymean basis as described by Fehr [20] was 054 when rust severity was measured in May and 060when measured in June The moderately high level of heritability will facilitate improvement throughthe selection of superior genotypes with low disease levels that can be inherited by the next generationThe clonally replicated genotypes used to calculate heritability were also used as checks in the fungicideexperiment As expected because these were genotypes with extreme phenotypes the susceptiblechecks exhibited more severe infection (Figure 1b)

Sustainability 2018 10 696 5 of 12Sustainability 2018 10 x FOR PEER REVIEW 5 of 12

Figure 1 Effect of fungicide application on silphium rust infection Treatments included Fungicide (Azoxystrobin (Heritagereg) (solid bars) and No treatment a water application of the same volume as the fungicide (diagonal line bars) Effect was measured as the percent rust severity on individual leaves Bars represent mean plusmn SE (a) Rust severity was measured in May June and July on experimental plants (b) Average infection of check genets from three individual plant replicates for each treatment Resistant checks included SI1100 SI1830 and SI1066Susceptible checks included SI-1619 SI591 and SI175 H annuus included HA-89 and HA-292 Mean comparisons were made using a Tukey HSD (honest significance difference) test

Stalks with meristem or complete necrosis occurred on average in 119 (SE = 17) of plants in the treated plots and 191 (SE = 32) in the control plots Interestingly rust severity in June was weakly to moderately but significantly correlated with meristem necrosis (rsup2 = 034 p lt 005) (Figure 2a) suggesting that rust caused by P silphii might have contributed although it was not the only cause of stem damage It has been previously predicted that Lygus lineolaris (Palisot de Beauvois Hemiptera Miridae) bugs cause meristem damage [26] because as they feed they secrete an enzyme that causes the necrosis and abscission of reproductive structures [27]

Significant negative correlations were observed between the rust response in June and the yield components including number (rsup2 = minus023 p lt 005) and weight (rsup2 = minus028 p lt 005) of capitula (Figure 2b) as well as the stem rust severity and total leaf weight (rsup2 = minus030 p lt 005) (Figure 2c) These results indicate two ways in which rust may limit yield in silphium first infection on the stem (meristem necrosis associated with rust damage) may limit the production of new leaves and thus reduce total leaf area of the plants and number of capitula additionally that infection on the leaves themselves may limit the current total CO2 uptake and thus carbohydrate availability for the growth of the capitula resulting in smaller capitula The number of capitula dry weight of leaves stems and

0

2

4

6

8

10

H annuus Resistant checks Susceptible checks

Rust

infe

ctio

n se

verit

y (

leaf

are

a)

Check genets

Fungicide No treatment

b

ab

a a a a

Figure 1 Effect of fungicide application on silphium rust infection Treatments included Fungicide(Azoxystrobin (Heritagereg) (solid bars) and No treatment a water application of the same volumeas the fungicide (diagonal line bars) Effect was measured as the percent rust severity on individualleaves Bars represent mean plusmn SE (a) Rust severity was measured in May June and July onexperimental plants (b) Average infection of check genets from three individual plant replicatesfor each treatment Resistant checks included SI1100 SI1830 and SI1066Susceptible checks includedSI-1619 SI591 and SI175 H annuus included HA-89 and HA-292 Mean comparisons were made usinga Tukey HSD (honest significance difference) test

Stalks with meristem or complete necrosis occurred on average in 119 (SE = 17) of plantsin the treated plots and 191 (SE = 32) in the control plots Interestingly rust severity in Junewas weakly to moderately but significantly correlated with meristem necrosis (r2 = 034 p lt 005)(Figure 2a) suggesting that rust caused by P silphii might have contributed although it was not theonly cause of stem damage It has been previously predicted that Lygus lineolaris (Palisot de BeauvoisHemiptera Miridae) bugs cause meristem damage [26] because as they feed they secrete an enzymethat causes the necrosis and abscission of reproductive structures [27]

Significant negative correlations were observed between the rust response in June and the yieldcomponents including number (r2 = minus023 p lt 005) and weight (r2 = minus028 p lt 005) of capitula(Figure 2b) as well as the stem rust severity and total leaf weight (r2 = minus030 p lt 005) (Figure 2c)These results indicate two ways in which rust may limit yield in silphium first infection on the stem(meristem necrosis associated with rust damage) may limit the production of new leaves and thusreduce total leaf area of the plants and number of capitula additionally that infection on the leavesthemselves may limit the current total CO2 uptake and thus carbohydrate availability for the growthof the capitula resulting in smaller capitula The number of capitula dry weight of leaves stems


and capitula were similar in the fungicide-treated and control plots (p = 007ndash025) Despite significantlyreducing infection in June when plants were at full bloom (Figure 1a) the effects of rust on yield couldnot be controlled by fungicide applications

Sustainability 2018 10 x FOR PEER REVIEW 6 of 12

capitula were similar in the fungicide-treated and control plots (p = 007ndash025) Despite significantly reducing infection in June when plants were at full bloom (Figure 1a) the effects of rust on yield could not be controlled by fungicide applications

Figure 2 Yield components with rust infection (a) Percent necrotic stalks from individuals differing in rust severity on the leaf (b) dry weight of heads (capitula) from individuals differing in rust response on the leaf (c) dry weight of leaves from individuals differing in rust infection severity on the stems Points were jittered to distinguish overlapping observations rsup2 represents the Pearsonrsquos coefficient of correlation

Rust negatively affected CO2 uptake (Figure 3a) while transpiration (E) was not dependent on the level of infection (Figure 3b) Net CO2 uptake decreased by approximately three-fold from a 01 to a 2 level of disease severity on the leaves and decreased approximately 50 from a 0 to a 1 level of rust severity (Figure 3a) We found that infection in a relatively small area of the leaf had a very large impact on the photosynthetic capacity of the entire leaf (Figure 3a) Because of the different response of assimilation rate (A) (assimilation decreased with rust) and transpiration (E) (transpiration was unaffected by rust Figure 3b) water use efficiency (WUE) followed a negative response curve similar to that of A as rust severity increased (Figure 3c) Some studies have found that CO2 uptake was reduced due to lower photosynthetic capacity in the leaf area between rust pustules [12] which was attributed to the reduced activity of Rubisco in the remaining healthy areas of the leaves [14] Leaf rust reduced net CO2 uptake at light saturation through reductions in gross photosynthesis rather than through increases in dark respiration rate in wheat [28] and barley [29] In silphium we do not know the cause of the reduction in CO2 uptake there could be a reduction in chlorophyll content a physical inhibition or shadowing effect of the rust lesions or a reduction in carboxylation We hypothesize that stomatal limitation is probably not the cause of the reduction in CO2 uptake since transpiration was not affected by rust severity this opens a new question for future research

Figure 2 Yield components with rust infection (a) Percent necrotic stalks from individuals differing inrust severity on the leaf (b) dry weight of heads (capitula) from individuals differing in rust responseon the leaf (c) dry weight of leaves from individuals differing in rust infection severity on the stemsPoints were jittered to distinguish overlapping observations r2 represents the Pearsonrsquos coefficientof correlation

Rust negatively affected CO2 uptake (Figure 3a) while transpiration (E) was not dependent on thelevel of infection (Figure 3b) Net CO2 uptake decreased by approximately three-fold from a 01 toa 2 level of disease severity on the leaves and decreased approximately 50 from a 0 to a 1 levelof rust severity (Figure 3a) We found that infection in a relatively small area of the leaf had a very largeimpact on the photosynthetic capacity of the entire leaf (Figure 3a) Because of the different responseof assimilation rate (A) (assimilation decreased with rust) and transpiration (E) (transpiration wasunaffected by rust Figure 3b) water use efficiency (WUE) followed a negative response curve similarto that of A as rust severity increased (Figure 3c) Some studies have found that CO2 uptake wasreduced due to lower photosynthetic capacity in the leaf area between rust pustules [12] which wasattributed to the reduced activity of Rubisco in the remaining healthy areas of the leaves [14] Leaf rustreduced net CO2 uptake at light saturation through reductions in gross photosynthesis rather thanthrough increases in dark respiration rate in wheat [28] and barley [29] In silphium we do not knowthe cause of the reduction in CO2 uptake there could be a reduction in chlorophyll content a physicalinhibition or shadowing effect of the rust lesions or a reduction in carboxylation We hypothesize thatstomatal limitation is probably not the cause of the reduction in CO2 uptake since transpiration wasnot affected by rust severity this opens a new question for future research


Figure 3 Instantaneous gas exchange from leaves with different levels of rust infection (a) Net CO2 assimilation rate (A) for fully expanded silphium leaves differing in severity of rust infection (b) transpiration (E) (c) water use efficiency (WUE) Each data point represents one leaf per plant Gas exchange was measured at noon (1200 am to 200 pm) on a sunny day on fully expanded leaves exposed to full-sun radiation using an infrared gas analyzer In (b) the relationship between E and rust severity was not significant therefore no trend line was fitted

DCM (dicloromethane) extracts a proxy for total resin content averaged approximately 85 of the dry weight in non-infected leaves (non-infected leaves and leaves with very low infection of 01 severity) of both susceptible and resistant plants (Figure 4) Leaves infected with a severity of ge1 accumulated more resin than non-infected leaves on the same plant (Figure 4) At the time when leaves were collected for resin analysis some of the younger leaves appeared uninfected while others were infected Presumably the difference in infection was caused by a latent period of disease development following humid microclimate conditions required for spore germination Terpenes such as resins and essential oils are common in members of the Asteraceae [22] most of which are known to have antibacterial antifungal and anti-herbivory effects [3031] Resin content is dependent on CN ratios and can be triggered by external stimuli such as resource availability herbivory [32] and disease infection [3334]

These results support the hypothesis that resins are involved in the response to rust but do not directly support the hypothesis that elevated resin content protects plants from infection One interpretation of this result is that Puccinia elicits plant resin production for its own benefit Maize biotrophic fungi have for example been shown to upregulate anthocyanin biosynthesis presumably to limit the substrates available for more effective defense responses [35] Another interpretation is that plants are triggered to synthesize resins after infection to limit the success of secondary infections [36] A final possibility is that the domestication process has resulted in lower baseline but more

Figure 3 Instantaneous gas exchange from leaves with different levels of rust infection (a) NetCO2 assimilation rate (A) for fully expanded silphium leaves differing in severity of rust infection(b) transpiration (E) (c) water use efficiency (WUE) Each data point represents one leaf per plantGas exchange was measured at noon (1200 am to 200 pm) on a sunny day on fully expanded leavesexposed to full-sun radiation using an infrared gas analyzer In (b) the relationship between E andrust severity was not significant therefore no trend line was fitted

DCM (dicloromethane) extracts a proxy for total resin content averaged approximately 85 ofthe dry weight in non-infected leaves (non-infected leaves and leaves with very low infection of 01severity) of both susceptible and resistant plants (Figure 4) Leaves infected with a severity of ge1accumulated more resin than non-infected leaves on the same plant (Figure 4) At the time when leaveswere collected for resin analysis some of the younger leaves appeared uninfected while others wereinfected Presumably the difference in infection was caused by a latent period of disease developmentfollowing humid microclimate conditions required for spore germination Terpenes such as resinsand essential oils are common in members of the Asteraceae [22] most of which are known to haveantibacterial antifungal and anti-herbivory effects [3031] Resin content is dependent on CN ratiosand can be triggered by external stimuli such as resource availability herbivory [32] and diseaseinfection [3334]

These results support the hypothesis that resins are involved in the response to rustbut do not directly support the hypothesis that elevated resin content protects plants frominfection One interpretation of this result is that Puccinia elicits plant resin production for itsown benefit Maize biotrophic fungi have for example been shown to upregulate anthocyaninbiosynthesis presumably to limit the substrates available for more effective defense responses [35]Another interpretation is that plants are triggered to synthesize resins after infection to limit the success


of secondary infections [36] A final possibility is that the domestication process has resulted in lowerbaseline but more plastic allocation to resins Higher resin content in wild compared to selected plantshas been observed in silphium [6] Likewise domesticated sunflowers produce fewer sesquiterpenelactone-containing trichomes than wild accessions although investment appeared to be constitutivenot plastic [37] Low resin production prior to infection may have made our selected populationsmore susceptible to rust although some rare individuals have resistance genes or physicalchemicaldefenses independent of resins When our breeding populations were small the population of rustwas also small In this environment the reallocation of resources towards sexual reproduction couldhave favored a more inducible resin response Although our results show that resin levels can increasemarkedly upon infection in this case the induced response may have been too small in magnitude ortoo late to reduce infection


plastic allocation to resins Higher resin content in wild compared to selected plants has been observed in silphium [6] Likewise domesticated sunflowers produce fewer sesquiterpene lactone-containing trichomes than wild accessions although investment appeared to be constitutive not plastic [37] Low resin production prior to infection may have made our selected populations more susceptible to rust although some rare individuals have resistance genes or physicalchemical defenses independent of resins When our breeding populations were small the population of rust was also small In this environment the reallocation of resources towards sexual reproduction could have favored a more inducible resin response Although our results show that resin levels can increase markedly upon infection in this case the induced response may have been too small in magnitude or too late to reduce infection

Figure 4 Resin content for non-infected and rust infected leaves from plants that were resistant and susceptible to rust Rust severity on infected leaves was ge10 for susceptible plants and between 01 and 10 for resistant plants Error bars represent the mean plusmn SE (F = 597 p lt 001) Mean comparisons were made using a Tukey HSD test DW is dry weight

4 Key Outcomes and Future Directions

As in many agriculturally significant crops we have verified that rust infection causes severe damage in silphium We found that leaf processes (gas exchange net CO2 uptake and water use efficiency) and productivity indicators (head number head weight and leaf weight) were reduced when silphium leaves and stems were more heavily infected by P silphii Although rust severity increased on average between May and June and even in July plants exhibited a wide range of damage A few individuals appeared to be resistant Many others appeared to be susceptible but able to limit or tolerate rust and still produce capitula and seeds These results are consistent with the existence within the silphium breeding population of both complete protection likely conferred by major-effect resistance genes (eg ldquoR-genesrdquo) and incomplete resistance genes conferred by multiple genes of partial effect [38] Further evaluation will be needed to determine the types of resistance in silphium which may be polygenic partial or growth stage-dependent With the high heritability observed for resistance to rust selecting for resistant plants is a promising strategy for decreasing rust infection in the population

We present preliminary evidence that leaf resin content increases when susceptible plants are infected with rust Infected leaves from plants with generally mild symptoms (resistant plants) do not show elevated resin content To understand these differences a deeper understanding of other defense mechanisms in the more resistant plants is needed Of particular interest is the potential presence of resistance genes and how those genes function to prevent infection The effect of resins in limiting rust and other pathogens or insect pests is an additional area of interest

Controlling rust using fungicides may be practical for particularly high-value plants (eg research populations) or to improve seedling survival and rate of establishment However even though the fungicide used in this experiment was effective at reducing rust severity it was ineffective

Figure 4 Resin content for non-infected and rust infected leaves from plants that were resistant andsusceptible to rust Rust severity on infected leaves was ge10 for susceptible plants and between 01and 10 for resistant plants Error bars represent the mean plusmn SE (F = 597 p lt 001) Mean comparisonswere made using a Tukey HSD test DW is dry weight


As in many agriculturally significant crops we have verified that rust infection causes severedamage in silphium We found that leaf processes (gas exchange net CO2 uptake and water useefficiency) and productivity indicators (head number head weight and leaf weight) were reducedwhen silphium leaves and stems were more heavily infected by P silphii Although rust severityincreased on average between May and June and even in July plants exhibited a wide range ofdamage A few individuals appeared to be resistant Many others appeared to be susceptible butable to limit or tolerate rust and still produce capitula and seeds These results are consistent with theexistence within the silphium breeding population of both complete protection likely conferred bymajor-effect resistance genes (eg ldquoR-genesrdquo) and incomplete resistance genes conferred by multiplegenes of partial effect [38] Further evaluation will be needed to determine the types of resistance insilphium which may be polygenic partial or growth stage-dependent With the high heritabilityobserved for resistance to rust selecting for resistant plants is a promising strategy for decreasing rustinfection in the population

We present preliminary evidence that leaf resin content increases when susceptible plants areinfected with rust Infected leaves from plants with generally mild symptoms (resistant plants) do notshow elevated resin content To understand these differences a deeper understanding of other defensemechanisms in the more resistant plants is needed Of particular interest is the potential presence ofresistance genes and how those genes function to prevent infection The effect of resins in limiting rustand other pathogens or insect pests is an additional area of interest


Controlling rust using fungicides may be practical for particularly high-value plants (eg researchpopulations) or to improve seedling survival and rate of establishment However even though thefungicide used in this experiment was effective at reducing rust severity it was ineffective at preventingyield losses We believe the fungicide application in our experiment was largely ineffective later in thegrowing season because the canopy structure of silphium makes it very difficult to achieve completeleaf coverage especially as the plants rapidly increase in number of nodes and plant height in JuneIt is possible that fungicides with different active ingredients or more complete coverage might bemore effective It appears that in the absence of total control P silphii can rapidly reproduce andre-infect new growth

41 Priorities for Achieving Future Progress

Taken together the leaf functional responses to P silphii appear to be non-linear such thatwhile extremely mild symptoms may prove to be economically acceptable intermediate andsevere symptoms are both associated with serious depression of CO2 uptake At the same timeleaf transpiration is not reduced suggesting that stomatal conductance remains high Combinedwith the elevated synthesis of resins the reduction of CO2 uptake means that infected leaves likelybecome net carbon sinks Increased susceptibility to other forms of stem and meristem damage furthercompound the effects of intermediate and severe infection

We have seen very little short-term silphium mortality due to rust infection and we have notfound evidence that rust causes severe lodging or sterility Some non-lethal plant diseases need tobe controlled primarily to reduce the production of spores (eg that could infect alternate hosts)to reduce the amount of toxins in food products or to improve the cosmetic appeal of crops toconsumers Silphium rust does not appear to infect sunflower and has not been reported to havealternate hosts outside of the genus Seeds are generally free from rust pustules meaning that grainqualityfood safety is not a primary concern

However the negative effects of even moderately severe rust on silphium carbon and waterbudgetsmdashand the challenge of controlling rust chemicallymdashmakes breeding for resistance a highpriority Silphium perfoliatum L (cup plant) is nearly immune at least to the populations of P silphiinaturally occurring in our breeding nurseries [39] (unpublished results) We have made interspecificcrosses between S perfoliatum and S integrifolium to determine if non-host resistance alleles can beintrogressed into S integrifolium populations We are also pursuing a multigenic ldquohorizontalrdquo resistanceapproach by including a leaf health score (primarily the absence of rust pustules) in our selection indexalong with seed and yield traits

42 Broader Implications

Perennial crops are likely to capture more atmospheric carbon and soil water than annual cropsby virtue of their longer growing season [40] This proposition underlies two central rationalesfor investing in the development of perennial grains (1) It makes perennial grains possiblethe extra carbon and water acquisition is necessary to support high allocation to harvested grainwhile simultaneously supporting additional plant ldquosinksrdquo including overwintering storage organs [40](2) This extra acquisition of carbon and water is acceptable or beneficial environmentally For exampleadditional assimilated carbon should end up being sequestered belowground helping to stabilizeatmospheric carbon dioxide levels [41] Water acquired from below the rooting zone of standard fieldcrops should stabilize agroecosystem productivity during short-term droughts [42]

The efficient use of both carbon dioxide and water by perennial crops is necessary for either ofthese rationales The evidence presented here suggests that foliar diseases can make a perennialplant highly inefficient with both acquired CO2 and H2O potentially undermining the veryrationale for pursuing a new perennial crop This finding illustrates the need for multidisciplinarydomesticationperennialization teams to monitor changes occurring as the consequence of geneticselection andor agroecosystem assembly [5]


A third rationale for perennial cropsmdashthat they are likely to be stress-tolerantmdashis based on theobservation that wild perennial relatives of crops are often sources of genes for resistance to bioticand abiotic stress [43] perhaps because perennials have a history of natural selection for persistencePerennial crops therefore could help farmers achieve goals of reduced dependence upon syntheticpesticides or other inputs While we observe that Silphium integrifolium indeed resists many abioticstresses and generalist herbivores [39] it is or has become highly susceptible to specialist pests anddiseases as shown here for the case of Puccinia silphii

Specialists by definition have few if any alternate hostsfoods and are therefore under strongnatural selection to overcome their host plantrsquos defenses Native prairie plants experience high ratesof infestation compared with exotic plants [44] We speculate that moving a wild plant from theforest or prairie may allow it to escape its enemies temporarily when initially evaluated at a researchstation However when grown in large numbers or in monocultures native plants may become evenmore vulnerable than they were in the wild as host abundance is correlated with prairie pathogenseverity [45] As with Silphium integrifolium switchgrass (Panicum virgatum L) another North Americannative perennial under domestication was relatively unaffected by diseases during early research butthe incidence of switchgrass rust (Puccinia emaculata) has increased and negatively impacted ethanolyield [46] In light of this we hypothesize that the yield of new perennial crops is more likely to bethreatened by specialist than by generalist agricultural pests when first brought into cultivation If thisis true the domestication of a perennial crop in its native range might be most efficient if aggressiveresearch for managing specialist pestspathogens were to precede investment in selection for classicdomestication traits even if few problems are initially observed Alternatively domestication could bedone outside the range of these specialists

Supplementary Materials The following are available online at wwwmdpicom2071-1050103696s1Figure S1 Rust scoring guide for silphium with pictorial examples

Acknowledgments We thank Eline van de Ven from the HAS University of Applied Sciences in the Netherlandsfor her substantial contribution in collecting and organizing data for the yield experiment We gratefullyacknowledge support from the Perennial Agriculture Project a joint project between The Land Institute and TheMalone Family Land Preservation Foundation

Author Contributions M Kathryn Turner Damian Ravetta and David Van Tassel conceived and designedthe experiments M Kathryn Turner and Damian Ravetta performed the experiments M Kathryn Turner andDamian Ravetta analyzed the data Damian Ravetta and David Van Tassel contributed reagentsmaterialsanalysistools M Kathryn Turner Damian Ravetta and David Van Tassel wrote the paper

Conflicts of Interest The authors declare no conflict of interest The Perennial Agriculture Project had no role inthe design of the study in the collection analyses or interpretation of data in the writing of the manuscript or inthe decision to publish the results

References

1 Gonzaacutelez-Paleo L Ravetta DA Relationship between photosynthesis water use and leaf structure indesert annual and perennial forbs differing in growth Photosynthetica 2018 56 in press

2 Jenks MA Joly R Peters PJ Rich PJ Axtell JD Ashworth EN Chemically Induced Cuticle MutationAffecting Epidermal Conductance to Water Vapor and Disease Susceptibility in Sorghum bicolor (1) MoenchPlant Physiol 1994 105 1239ndash1245 [CrossRef] [PubMed]

3 Sevillano M Del Ci M Drsquoambrogioj A Pastranai CP Sierrai E Resistance to rust caused by foliarpubescence in Argentinean common bean cultivars Ann Rep Bean Improv Coop 1997 40 108ndash109

4 Hol WHG Van Veen JA Pyrrolizidine alkaloids from Senecio jacobaea affect fungal growth J Chem Ecol2002 28 1763ndash1772 [CrossRef] [PubMed]

5 Van Tassel DL Albrecht KA Bever JD Boe AA Brandvain Y Crews TE Gansberger MGerstberger P Gonzaacutelez-Paleo L Hulke BS et al Accelerating Domestication An Opportunity toDevelop New Crop Ideotypes and Breeding Strategies Informed by Multiple Disciplines Crop Sci 2017 571274 [CrossRef]


6 Vilela AE Gonzaacutelez-Paleo L Turner MK Peterson KE Ravetta DA Crews TE Van Tassel DLProgress and observations during the early cultivation and domestication of Silphium integrifolium asa future perennial oilseed sunflower substitute Sustainability 2018 10 638 [CrossRef]

7 Arthur JC Manual of the Rusts in United States and Canada Purdue research foundation Lafayette IN USA 19348 Bolton MD Kolmer JA Garvin DF Wheat leaf rust caused by Puccinia triticina Mol Plant Pathol 2008

9 563ndash575 [CrossRef] [PubMed]9 Caldwell R Kraybill H Sullivan J Compton L Effect of leaf rust (Puccinia triticina) on yield physical

characters and composition of winter wheats J Agric Res 1934 48 1049ndash107110 Dyck PL Lukow OM The genetic analysis of two interspecific sources of leaf rust resistance and their

effect on the quality of common wheat Can J Plant Sci 1988 68 633ndash639 [CrossRef]11 Everts KL Leath S Finney PL Impact of Powdery Mildew and Leaf Rust on Milling and Baking Quality

of Soft Red Winter Wheat Plant Dis 2001 85 423ndash429 [CrossRef]12 Scholes JD Farrar JF Increased rates of photosynthesis in localized regions of a barley leaf infected with

brown rust New Phytol 1986 104 601ndash612 [CrossRef]13 Magyarosy AC Schurmann P Buchanan BB Effect of Powdery Mildew Infection on Photosynthesis by

Leaves and Chloroplasts of Sugar Beets1 Plant Physiol 1976 57 486ndash489 [CrossRef] [PubMed]14 Bassanezi RB Amorim L Filho AB Berger RD Gas Exchange and Emission of Chlorophyll Fluorescence

during the Monocycle of Rust Angular Leaf Spot and Anthracnose on Bean Leaves as a Function of theirTrophic Characteristics J Phytopathol 2002 150 37ndash47 [CrossRef]

15 Lasko A Pratt C Pearson RC Pool RM Seem RC Welser MJ Photosynthesis transpirationand water use efficiency of mature grape leaves infected with Uncinula necator (powdery mildew)Phytopathology 1982 72 232ndash236

16 Paul ND Ayres PG Effects of rust and post-infection drought on photosynthesis growth and waterrelations in groundsel Plant Pathol 1984 33 561ndash569 [CrossRef]

17 Gulya T Venette R Venette JR Lamey HA Sunflower Rust NDSU Extension Service Fargo ND USA 199018 Sackston WE Studies on sunflower rust III Occurrence distribution and significance of races of Puccinia

helianthi Schw Can J Bot 1962 40 1449ndash1458 [CrossRef]19 Friskop A Markell SG Khan M North Dakota Field Crop Fungicide GuidemdashPP622mdashPublications

Available online httpswwwagndsuedupublicationslanding-pagescropsnorth-dakota-field-crop-fungicide-guide-pp-622 (accessed on 8 December 2017)

20 Fehr W Principles of Cultivar Development Volume 1 Theory and Technique Macmillion Publishing Co AmesIA USA 1987

21 Ravetta DA Goffman F Pagano E McLaughlin SP Grindelia chiloensis resin and biomass production inits native environment Indus Crops Prod 1996 5 235ndash238 [CrossRef]

22 Langenheim JH Plant Resins Chemistry Evolution Ecology and Ethnobotany Timber Press Portland ORUSA 2003

23 R Core Team R A Language and Environment for Statistical Computing R Core Team Vienna Austria 201424 Gold RE Mendgen K Rust Basidiospore Germlings and Disease Initiation In The Fungal Spore and Disease

Initiation in Plants and Animals Springer Boston MA USA 1991 pp 67ndash9925 Parmelee JA The Autoecious species of Puccinia on Heliantheae in North America Can J Bot 1967 45

2267ndash2327 [CrossRef]26 Prasifka JR Mallinger RE Hulke BS Larson SR Van Tassel D PlantndashHerbivore and Plant-Pollinator

Interactions of the Developing Perennial Oilseed Crop Silphium integrifolium Environ Entomol 2017 461339ndash1345 [CrossRef] [PubMed]

27 Strong FE Physiology of Injury Caused by Lygus hesperus J Econ Entomol 1970 63 808ndash814 [CrossRef]28 Carretero R Bancal MO Miralles DJ Effect of leaf rust (Puccinia triticina) on photosynthesis and

related processes of leaves in wheat crops grown at two contrasting sites and with different nitrogen levelsEur J Agron 2011 35 237ndash246 [CrossRef]

29 Owera S Farrar J Whitbread R Growth and photosynthesis in barley infected with brown rustPhysiol Plant Pathol 1981 18 79ndash90 [CrossRef]

30 Joseacute Abad M Miguel Bedoya L Bermejo P Essential Oils from the Asteraceae Family Active againstMultidrug-Resistant Bacteria In Fighting Multidrug Resistance with Herbal Extracts Essential Oils and TheirComponents Elsevier San Diego CA USA 2013 pp 205ndash221


31 Habermehl G Fliegner W Terpenes and their Biological Relevance In Studies in Natural Products ChemistryAtta-ur-Rahman Ed Elsevier Science Amsterdam The Nederland 1998 pp 3ndash24

32 Stout MJ Host-Plant Resistance in Pest Management In Integrated Pest Management Elsevier San DiegoCA USA 2014

33 Salomon MV Purpora R Bottini R Piccoli P Rhizosphere associated bacteria trigger accumulationof terpenes in leaves of Vitis vinifera L cv Malbec that protect cells against reactive oxygen speciesPlant Physiol Biochem 2016 106 295ndash304 [CrossRef] [PubMed]

34 Yoshitomi K Taniguchi S Tanaka K Uji Y Akimitsu K Gomi K Rice terpene synthase 24 (OsTPS24)encodes a jasmonate-responsive monoterpene synthase that produces an antibacterial γ-terpinene againstrice pathogen J Plant Physiol 2016 191 120ndash126 [CrossRef] [PubMed]

35 Tanaka S Brefort T Neidig N Djamei A Kahnt J Vermerris W Koenig S Feussner K Feussner IKahmann R A secreted Ustilago maydis effector promotes virulence by targeting anthocyanin biosynthesisin maize eLife 2014 3 e01355 [CrossRef] [PubMed]

36 Zeneli G Krokene P Christiansen E Krekling T Gershenzon J Methyl jasmonate treatment of matureNorway spruce (Picea abies) trees increases the accumulation of terpenoid resin components and protectsagainst infection by Ceratocystis polonica a bark beetle-associated fungus Tree Physiol 2006 26 977ndash988[CrossRef] [PubMed]

37 Rowe HC Ro D Rieseberg LH Response of Sunflower (Helianthus annuus L) Leaf Surface Defenses toExogenous Methyl Jasmonate PLoS ONE 2012 7 e37191 [CrossRef] [PubMed]

38 Van der Plank JE Disease Resistance in Plants Academic Press Orlando FL USA 196839 Van Tassel DL The Land Institute Salina KS USA Personal communication 201740 DeHaan LR Van Tassel DL Cox TS Perennial grain crops A synthesis of ecology and plant breeding

Renew Agric Food Syst 2005 20 5ndash14 [CrossRef]41 Crews T Rumsey B What Agriculture Can Learn from Native Ecosystems in Building Soil Organic Matter

A Review Sustainability 2017 9 578 [CrossRef]42 Glover JD Reganold JP Cox CM Plant perennials to save Africarsquos soils Nature 2012 489 359ndash361

[CrossRef] [PubMed]43 Glover JD Reganold JP Bell LW Borevitz J Brummer EC Buckler ES Cox CM Cox TS

Crews TE Culman SW et al Agriculture Increased food and ecosystem security via perennial grainsScience 2010 328 1638ndash1639 [CrossRef] [PubMed]

44 Han X Dendy SP Garrett KA Fang L Smith MD Comparison of damage to native and exotic tallgrassprairie plants by natural enemies Plant Ecol 2008 198 197ndash210 [CrossRef]

45 Mitchell CE Tilman D Groth JV Effects of grassland plant species diversity abundance and compositionon foliar fungal disease Ecology 2002 83 1713ndash1726 [CrossRef]

46 Sykes VR Allen FL Mielenz JR Stewart CN Windham MT Hamilton CY Rodriguez M Yee KLReduction of Ethanol Yield from Switchgrass Infected with Rust Caused by Puccinia emaculata BioEnergy Res2016 9 239ndash247 [CrossRef]

copy 2018 by the authors Licensee MDPI Basel Switzerland This article is an open accessarticle distributed under the terms and conditions of the Creative Commons Attribution(CC BY) license (httpcreativecommonsorglicensesby40)

Introduction

Materials and Methods

Susceptible and Resistant Plant Materials

Fungicide Experiment

Leaf Resin Content

Results and Discussion

Key Outcomes and Future Directions

Priorities for Achieving Future Progress

Broader Implications

References


long-distance dispersing urediniospore stages of macrocyclic rusts [7] It has been the most prevalentdisease in introductory plantings and breeding plots in central Kansas although powdery mildew(caused by Erysiphe cichoracearum) downy mildew (caused by Plasmopara halstedii) and stem spotting(caused by Alternaria alternate) have also been observed Between 2003 and 2013 there were very fewpests or diseases observed in silphium research plantings in Kansas The low initial levels of rustincidence may have been due to limited local sources of inoculum when the populations of silphiumwere small in research plantings and inoculum was spreading from dispersed native stands In 2014high moisture promoted the spread of rust from a densely planting stand of silphium to many ofthe other plots By 2015 many silphium plants failed to flower or produced very small late flowersSome of these individuals had necrosis on the apical meristem or entire length of the stalk and othershad severely stunted plant height These symptoms have persisted in subsequent years and promptedinterest in determining the impact of rust on the yield and physiology of silphium

Rust-causing pathogens are detrimental to biomass production and grain yield acrossmany economically significant crops Puccinia triticina Eriks which causes leaf rust in wheat(Triticum aestivum) durum wheat (T turgidum L var durum) cultivated emmer wheat (T dicoccon)and wild emmer wheat (T dicoccoides) Aegilops speltoides goatgrass (Ae cylindrica) and triticale(X Triticosecale) [8] reduces the photosynthetic capacity of the flag leaf and lower leaves as it utilizesassimilates that would be translocated to developing grain resulting in lower seed set and smallerkernel weight [8] The quality of the grain can also be affected Decreased protein content andincreased yellow pigmentation have been observed in infected grain [8ndash10] but vary by genotype andenvironment [11] Changes in photosynthesis due to foliar pathogens have been reported across a rangeof crop species including leaf rust (Puccinia hordei Otth) in barley (Hordeum vulgare L) [12] powderymildew (Eysiphe polygoni DC) in sugar beet (Beta vulgaris L) [13] and rust (Uromyces appendiculatus)angular leaf spot (Phaeoisariopsis griseola (Sacc) Ferr) and anthracnose (Colletotrichum lindemuthianum)in common bean (Phaseolus vulgaris L) [14] Besides reduction in photosynthesis infections cancause other physiological changes such as reduced water use efficiency as observed in powderymildew (Uncinula necator) in grape (Vitis vinefera and V labruscana) [15] and powdery mildew(Puccinia lagenophorae) in groundsel (Senecio vulgaris) under drought conditions [16]

Our objectives here were to determine the impact of silphium rust on plant yield and leaf functionWe compared the rust infection of individuals in a population of plants selected for yield and relatedthis to chemical changes at the leaf level We also estimated heritability for rust resistance and testedthe effect of a fungicide treatment on rust severity and yield

2 Materials and Methods

21 Susceptible and Resistant Plant Materials

We adapted the Puccinia helianthus disease scales developed by Gulya (1990) [17] and Sackston(1962) [18] to quantify P silphii percent severity (proportion of the total leaf area infected) and response(a measure of pustule size and vigor) on the leaves and stems of silphium plants The pictorial scalesfor severity and response (Supplementary Materials Figure S1) were used in July 2015 to identifyindividuals with high and low disease levels which were used as susceptible and resistant checksrespectively The determination of resistance and susceptibility was based on our observations ofinfection caused by naturally occurring pathogen populations in our breeding nurseries We arenot aware of any description of P silphii races differential reaction to various isolates or silphiumgenotypes evaluated for resistance to P silphii Resistant plants (le1 severity of infection on the leaves)included SI1100 SI1066 and SI1830 Susceptible plants (gt2 leaf severity) included SI591 SI1619and SI175 In 2015 very few flowering heads were produced by SI1066 SI1830 SI1619 and SI175These individual genets were identified from a large breeding nursery of over 7000 genets from100 half-sib families planted approximately 1 m from each other in 2013 Rust symptoms were seenon the edges of this plot in 2014 and by 2015 the vast majority of plants in the nursery were infected




















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Effect of Puccinia silphii on Yield Components and Leaf ...the domestication of wild plants has been...

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